Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum

1981 ◽  
Vol 17 (3) ◽  
pp. 197-211 ◽  
Author(s):  
Ben A. Murray ◽  
Lisa D. Yee ◽  
William F. Loomis
Keyword(s):  
Dictyostelium Discoideum ◽  
Intercellular Adhesion ◽  
Contact Sites ◽  
Immunological Analysis

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum

1983 ◽  
Vol 3 (5) ◽  
pp. 863-870
Author(s):  
B A Murray ◽  
H L Niman ◽  
W F Loomis
Keyword(s):  
Monoclonal Antibody ◽  
Dictyostelium Discoideum ◽  
Posttranslational Modification ◽  
Rabbit Antiserum ◽  
Intercellular Adhesion ◽  
Blocking Effect ◽  
Antigenic Determinants ◽  
Membrane Glycoprotein ◽  
Developmentally Regulated ◽  
Contact Sites

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

scholarly journals Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

1983 ◽  
Vol 3 (5) ◽  
pp. 863-870 ◽  
Author(s):  
B A Murray ◽  
H L Niman ◽  
W F Loomis
Keyword(s):  
Monoclonal Antibody ◽  
Dictyostelium Discoideum ◽  
Posttranslational Modification ◽  
Rabbit Antiserum ◽  
Intercellular Adhesion ◽  
Blocking Effect ◽  
Antigenic Determinants ◽  
Membrane Glycoprotein ◽  
Developmentally Regulated ◽  
Contact Sites

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

scholarly journals Interaction of integrins alpha 3 beta 1 and alpha 2 beta 1: potential role in keratinocyte intercellular adhesion.

1993 ◽  
Vol 120 (2) ◽  
pp. 523-535 ◽  
Author(s):  
B E Symington ◽  
Y Takada ◽  
W G Carter
Keyword(s):  
Potential Role ◽  
Intercellular Adhesion ◽  
Epidermal Cells ◽  
Intercellular Contact ◽  
In Vitro Experiments ◽  
Selective Binding ◽  
Contact Sites ◽  
Do So

The colocalization of integrins alpha 2 beta 1 and alpha 3 beta 1 at intercellular contact sites of keratinocytes in culture and in epidermis suggests that these integrins may mediate intercellular adhesion (ICA). P1B5, an anti-alpha 3 beta 1 mAb previously reported to inhibit keratinocyte adhesion to epiligrin, was also found to induce ICA. Evidence that P1B5-induced ICA was mediated by alpha 2 beta 1 and alpha 3 beta 1 was obtained using both ICA assays and assays with purified, mAb-immobilized integrins. Selective binding of alpha 2 beta 1-coated beads to epidermal cells or plate-bound alpha 3 beta 1 was observed. This binding was inhibited by mAbs to integrin alpha 3, alpha 2, or beta 1 subunits and could be stimulated by P1B5. We also demonstrate a selective and inhibitable interaction between affinity-purified integrins alpha 2 beta 1 and alpha 3 beta 1. Finally, we show that expression of alpha 2 beta 1 by CHO fibroblasts results in the acquisition of collagen and alpha 3 beta 1 binding. Binding to both of these ligands is inhibited by P1H5, an anti-alpha 2 beta 1 specific mAb. Results of these in vitro experiments suggest that integrins alpha 2 beta 1 and alpha 3 beta 1 can interact and may do so to mediate ICA in vivo. Thus, alpha 3 beta 1 mediates keratinocyte adhesion to epiligrin and plays a second role in ICA via alpha 2 beta 1.

The effects of inhibition of protein glycosylation on the aggregation of Dictyostelium discoideum

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 229-248
Author(s):  
Charles John McDonald ◽  
Jeffrey Sampson
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Total Protein ◽  
Subsequent Development ◽  
Protein Glycosylation ◽  
Normal Process ◽  
Contact Sites ◽  
Cyclic Amp Phosphodiesterase

At concentrations greater than 10 µg ml−1 tunicamycin inhibited the incorporation of [3H]mannose into glycoproteins during the early phase of development in Dictyostelium discoideum, however, total protein synthesis was unaffected. Tunicamycin also interfered with the normal process of aggregation. In its presence small aggregates were observed at the time of normal aggregation, but amoebae failed to aggregate completely and subsequent development was inhibited. Inhibition of normal aggregation by tunicamycin was found to be reversible. The appearance of cell-associated and secreted cyclic AMP phosphodiesterase and cell-surface contact sites A was prevented by tunicamycin but cell surface cyclic AMP receptor activity developed normally in its presence. Tunicamycin also prevented amoebae from acquiring the ability to chemotact toward cyclic AMP. Addition of exogenous cyclic AMP phosphodiesterase restored the ability of amoebae to chemotact toward cyclic AMP in the presence of tunicamycin. Our data suggest that the primary block in aggregation caused by tunicamycin results from the inhibition of expression of active cyclic AMP phosphodiesterase.

Physiological and biochemical characterization of aggregation-deficient mutants of Dictyostelium discoideum: detection and response to exogenous cyclic AMP

1978 ◽  
Vol 24 (4) ◽  
pp. 455-465 ◽  
Author(s):  
E. K.-L. Lo ◽  
M. B. Coukell ◽  
A. S. Tsang ◽  
J. L. Pickering
Keyword(s):  
Dictyostelium Discoideum ◽  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Chemotactic Response ◽  
Bacterial Suspension ◽  
Camp Phosphodiesterase ◽  
Contact Sites ◽  
Solid Substratum ◽  
Camp Receptor ◽  
Physiological And Biochemical

Forty independently isolated aggregation-deficient (Agg−) mutants of Dictyostelium discoideum, which have been partially characterized genetically, were examined for the ability to respond chemotactically to exogenous cAMP and for certain cellular and extracellular activities thought to be involved in this process. Only five of the mutants failed to produce a statistically significant chemotactic response to cAMP. When shaken in buffer, most of the Agg− mutants produced reduced levels of the cAMP-binding sites and the cell-bound cAMP phosphodiesterase reported to be components of a cell-surface cAMP receptor involved in the chemotactic response. In addition, most of the mutants failed to form contact sites A and were unable to develop on water agar plates. When the mutants were pulsed with 100 nM cAMP, 36 of 40 mutants produced detectable contact sites A and three-quarters of these strains underwent further development when placed on a solid substratum. Treatment with cAMP pulses also stimulated certain mutants to form higher levels of cAMP receptors. These observations suggest that the impaired differentiation of the plasma membrane of many Agg− mutants is due, at least in part, to the abnormal synthesis and (or) secretion of cAMP by these strains. When grown in bacterial suspension, all 40 of the mutants produced extracellular phosphodiesterase (ePD) activity, and all but 8 of the mutants secreted detectable amounts of the ePD inhibitor. In general, Agg− strains carrying mutations at the same aggregation locus (aggF–J) exhibited very similar physiological and biochemical properties.

Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum

1983 ◽  
Vol 60 (1) ◽  
pp. 251-266
Author(s):  
C.M. Chadwick ◽  
D.R. Garrod
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Protein A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Live Cells ◽  
Cell Contact ◽  
Vegetative Cells ◽  
Contact Sites

Polyspecific antibodies were raised against vegetative cells of Dictyostelium discoideum, strain Ax2. Monovalent (Fab') fragments of antibodies CMC 1, 5, 7 and 12 blocked completely the cohesion of vegetative cells. Antibody CMC 1 was studied in detail. The Fab' of this blocked the cohesion of aggregation-competent cells by 40%. It also caused some loss of cell contact in aggregation streams. In so doing the contacts that remained were mostly at the ends of the cells. Immunofluorescence showed that CMC 1 Fab' bound to both the cytoplasm and the surface of fixed cells. It also bound to the surface of live cells. A control (N Fab') also bound to the cell surface but did not block vegetative cell cohesion. An extract of vegetative cells was obtained using the detergent Triton X-100. D. discoideum proteins were immunoprecipitated from this extract using protein A-Sepharose and CMC 1 immunoglobulin G (IgG). These immobilized proteins absorbed the cohesion-blocking activity of CMC 1 Fab'. About 30 proteins were obtained when the Triton-soluble fraction was immunoprecipitated with IgG of CMC 1, 5, 7 and 12. Five of these were found to be cell surface proteins by the technique of lactoperoxidase-catalysed radio-iodination. These proteins had molecular weights of 178 000, 166 000, 126 000 and 64 000. CMC 12 IgG immunoprecipitated an additional cell surface protein of 46 000 molecular weight. Slices of polyacrylamide gel containing each of the five proteins identified as possible contact sites were fixed, washed and incubated with CMC 1 Fab'. Gel that contained protein of 178 000, 166 000 and 64 000 molecular weight had no effect on the activity of CMC 1 Fab'. However, Fab' that had been incubated with gel containing protein of 126 000 molecular weight no longer blocked cell cohesion.

Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA

1988 ◽  
Vol 8 (8) ◽  
pp. 3458-3466
Author(s):  
A E Early ◽  
J G Williams ◽  
H E Meyer ◽  
S B Por ◽  
E Smith ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Protein A ◽  
Surface Glycoprotein ◽  
Specific Gene ◽  
Mrna Accumulation ◽  
Cell Surface Glycoprotein ◽  
Repeat Elements ◽  
Contact Sites ◽  
C Terminus

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.

scholarly journals Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

1988 ◽  
Vol 8 (8) ◽  
pp. 3458-3466 ◽  
Author(s):  
A E Early ◽  
J G Williams ◽  
H E Meyer ◽  
S B Por ◽  
E Smith ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Protein A ◽  
Surface Glycoprotein ◽  
Specific Gene ◽  
Mrna Accumulation ◽  
Cell Surface Glycoprotein ◽  
Repeat Elements ◽  
Contact Sites ◽  
C Terminus

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.

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